Protocol for olefin metathesis reactions of hydrophobic substrates performed in aqueous emulsion with mechanical stirring or with microwaves support

Summary In the presented protocol, we describe the olefin metathesis of hydrophobic substrates in water emulsions using ruthenium catalysts in the presence of air. We detail the testing of mechanical foaming for emulsification and the use of microwave heating to optimize metathesis reaction efficiency. By utilizing relatively low catalyst loading and ensuring simple product isolation, the steps outlined in this protocol extend known methods for the aqueous metathesis techniques. For complete details on the use and execution of this protocol, please refer to Tyszka-Gumkowska et al. (2022).


SUMMARY
In the presented protocol, we describe the olefin metathesis of hydrophobic substrates in water emulsions using ruthenium catalysts in the presence of air. We detail the testing of mechanical foaming for emulsification and the use of microwave heating to optimize metathesis reaction efficiency. By utilizing relatively low catalyst loading and ensuring simple product isolation, the steps outlined in this protocol extend known methods for the aqueous metathesis techniques. For complete details on the use and execution of this protocol, please refer to Tyszka-Gumkowska et al. (2022).

BEFORE YOU BEGIN
Olefin metathesis reaction has become one of the most convenient methods for the construction of carbon-carbon double bonds. Mostly due to the discovery of robust and active ruthenium catalysts this transformation has found wide applications in academia and industry (Grela, 2014;Ogba et al., 2018). As in other organometallic transformations, metathesis reactions usually require carefully dried and degassed aromatic or chlorinated solvents to prevent catalyst decomposition and side products formation. Unfortunately, the described conditions are far from the ideal Green Chemistry protocols, which is a serious limitation to the even more ubiquitous application of metathesis reactions (Clavier et al., 2007). To address this issue, scientific community attempt several trials to perform this reaction under aqueous conditions, since water represents the major green solvent: cheap, easily available, and non-toxic.
The most straightforward strategy to achieve this goal was to perform reactions 'on water' in a heterogenous system with water insoluble metathesis catalysts, taking advantage of the hydrophobic effect that accelerates the reaction rate (Davis and Sinou, 2002). On the other hand, the addition of various amphiphilic substances (surfactants) allowed for effective olefin metathesis 'in water'. These additives lead to the formation of micelles capable of accommodating water-insoluble substrate(s) along with a metathesis catalyst and ensure an effective course of the metathesis reaction (Lipshutz et al., 2008;Lipshutz and Ghorai, 2010).
A similar strategy involved specially designed catsurfs (catalyst+surfactant) -catalysts able to acting simultaneously as initiators and surfactants (Gawin et al., 2010). Recently, several water-soluble catalysts have also been presented, gained by incorporation of ionic tags or PEGs into classical catalysts structure (Hong and Grubbs, 2006;Olszewski et al., 2020;Skowerski et al., 2012;Wang et al., 2015). Nevertheless, relatively high loading of these tailor-made catalysts is usually required.
Instead of using surfactants and specialized catalysts for olefin metathesis, a similar performance can be obtained by ultrasonication of a lipophilic substrate and a commercially available hydrophobic catalyst in an emulsion system (Gułajski et al., 2008(Gułajski et al., , 2019. Using this protocol, the hydrophobic catalyst and substrates are encapsulated in small droplets of reaction media, allowing the metathesis reaction to proceed smoothly. Herein, we present our attempt to perform olefin metathesis reactions under aqueous conditions, stressing out the practical aspects of this process performed on a larger scale, when one needs to consider not only the chemical but also the economical parameters of the transformation. In this respect, it seems reasonable to us to use water as a diluent to suspend the reactants, ensuring convenient stirring and heat transfer. To probe the usefulness of the developed conditions on the manufacturing of fine chemicals, we decided to study lipophilic liquid substrates in olefin metathesis using water as a reaction medium. These conditions offer a convenient tool for conducting olefin metathesis under more environmentally and user-friendly conditions (Tyszka-Gumkowska et al., 2022).

Preparation of FixCatBARF catalyst
Timing: 3 h These steps describe preparation of FixCatBARF catalyst, by ion exchange reaction between NaBARF and FixCat catalyst (Scheme 1). Reaction should be performed using Schlenk line equipment consisting of two neck round bottom flask or Schlenk flask with a vacuum manifold which is connected to a vacuum pump, and an inert gas manifold which is connected to a source of commercially available and high quality argon (HiQ Argon 6.0). All reaction steps should be carried out in a well ventilated fume hood.
1. Evacuate the oven-dried Schlenk flask (volume 100 mL) for at least 15 min and backfill it with argon (flow rate $4 L/min, pressure 0.2 bar). 2. Under argon atmosphere add NaBARF (250 mg, 0.282 mmol) to the Schlenk flask, carefully evacuate and backfill the flask with argon three-times more. 3. Under argon atmosphere add 70 mL of anhydrous DCM.
Note: NaBARF will not be fully dissolved in DCM.

After 15 min of stirring add commercially available FixCat catalysts in one portion under argon
atmosphere (1.0 equiv., 250 mg, 0.282 mmol) and stir for 1 h at $20 C-25 C.

KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

MATERIALS AND EQUIPMENT
CRITICAL: Ruthenium complexes are potentially mutagenic and toxic compounds. 9-decen-1-ol, crotonaldehyde, and 6-chlorohexene are flammable, hazards for environment and irritating reagents, when 4-methyl-1-pentene is also highly volatile. Avoid contact of these substances with skin and eyes, and do not inhale or ingest them. Use personal protective equipment such as lab coat, goggles and gloves. All reaction setups should be carried out in well ventilated fume hood.
Alternatives: Reagents can be replaced by those obtained from other suppliers, as long as they are of the same quality and purity as those used in presented protocols; except fiveneck glass reactor for synthesis of 9-chloro-2-methylnon-4-ene round bottom flask closed with septum can be used; milk frother can be replaced with mechanical stirrer or strong magnetic stirrer; Kugel Rohr distillation apparatus can be replaced with classical glassware for distillation under reduce pressure; CEM Discover SP for synthesis of 11-hydroxyundec-2- The resources listed in the below table were based on our experience. Generally, the chemicals and resources can be purchased from any reliable commercial sources and do not need to be limited to those listed in our table.

Timing: 5.5 h
These steps describe the synthesis of 9-chloro-2-methylnon-4-ene by the metathesis reaction of 6-chlorohexene and 4-methyl-1-pentene using UltraCat as a catalyst (Scheme 2). Moreover, in this protocol, milk frother is used for emulsification of the reaction mixture: water, ethyl acetate, lipophilic substrates, and catalyst. All reaction steps should be carried out in well ventilated fume hood.
1. Preparation of substrates and solvents ( Figure 1): a. measure out substrates with a PP/PE syringes tipped with a single-use SS/PE needles 0.80 3 120 mm: 6.55 mL of 6-chlorohexene and 18.80 mL of 4-methyl-1-pentene; b. weigh out 801 mg of UltraCat catalyst in the glass vial. c. measure out 45 mL of distilled water and 12 mL of ethyl acetate with the cylinder; d. weigh out 474 mg of SnatchCat scavenger in the glass vial.
Scheme 2. Synthesis of 9-chloro-2-methylnon-4-ene by metathesis reaction in emulsion system using milk frother Note: The catalyst can be weighed out in air, but it should be stored under argon in the refrigerator at approximately 4 C. In these studies, it was possible for us to use a lower quality UltraCat catalyst (60% purity) as it gave identical result as the freshly purified one.
2. Set up the reaction ( Figure 2): a. prepare the reactor (volume 100 mL) by placing the tip of the milk frother in one of the necks; b. connect the upper and lower parts of the glass reactor: lubricate the ground glass part with hydrocarbon-based grease and tighten them with a chain clamp; c. pour water into the reactor through one of the necks with the help of a funnel and heat it to 40 C; d. after reaching the appropriate temperature, inject into the reactor the 6-chlorohexene and 4-methyl-1-pentene prepared in syringes through the neck of the reactor ended with a rubber septum; e. turn on the milk frother to form an emulsion; f. once the reaction has reached the desired temperature (40 C), add the UltraCat catalyst and pour ethyl acetate into the reactor; troubleshooting 1; g. continue stirring for 5 h at 40 C.
Pause point: Once the temperature and stirring mixer have stabilized, the reaction is ready to run.
Optional: Presented five-neck glass reactor can be replace by round bottom flask (volume 100 mL) with septum. Except milk frother some mechanical or magnetic stirrer can be used.

Timing: 2 h
These steps describe the purification of the reaction mixture by removing the catalyst and distilling the crude product. All these steps should be carried out in well ventilated fume hood.
3. After completion of the reaction, transfer the mixture to a 250 mL separatory funnel and separate the aqueous phase (bottom layer) from the organic one (top layer). Troubleshooting 2 and 3. 4. Place aqueous phase in beaker. 5. Transfer the organic phase to an Erlenmeyer flask. 6. Put the aqueous phase back into the separatory funnel, add 10 mL of ethyl acetate, shake the separatory funnel vigorously and, after separating the two phases, transfer organic phase to suitable Erlenmeyer flasks (Figure 3).

OPEN ACCESS
Note: Repeat this step two more times or until all product will be transfer to organic phase. Transfer of the product to organic layer can be monitored using thin layer chromatography (silica gel, hexane:ethyl acetate, 9:1).
7. Discard the aqueous phase in the appropriate waste container. 8. Add 474 mg of SnatchCat scavenger to the organic fractions collected in the Erlenmeyer flask and stir this mixture for at least 30 min. 9. Add drying agent (MgSO 4 or Na 2 SO 4 ), gently stir it and filter into a round bottom flask. Troubleshooting 4. 10. Remove $95% of the solvent using rotary evaporator (35 C, 130 mbar, $15 min). 11. Prepare a silica gel pad ($20 cm length, 3 cm diameter) and place the crude product on top, washing the round bottom flask with a small amount of hexane ( Figure 4). 12. Carefully wash the product off the silica gel pad with hexane under vacuum filtration.
CRITICAL: The dark brown residue of the catalyst after filtration should remain on top of the silica gel pad. The operation should be repeated if the catalyst residues penetrate too much into the silica gel layer and color the crude product solution. Any catalyst residues left in the crude product can cause its isomerization or decomposition.
13. Remove the solvent on a rotary evaporator (35 C, 130 mbar, $20 min).   Note: Do not use a high vacuum pump for the long-term drying of the crude product due to its volatility.
15. Purify the crude product by distillation ( Figure 5): a. set up the parameter on Kugel Rohr distillation apparatus to distill out the unreacted starting material: 100 C, 50 rpm and 50 mbars on vacuum pump; troubleshooting 5. b. collect the first fraction in the bulb; c. place a new bulb in the apparatus, raise the temperature to 160 C, and collect another fraction; d. place new bulb in the apparatus, raise the temperature to 200 C, and collect the pure product; e. leave a higher boiling yellow residue in the round bottom flask.
Optional: Kugel Rohr apparatus can be replaced by standard laboratory glassware for distillation under vacuum.
16. Transfer the distilled product to the fresh round bottom flask (previously tared or weighed), wash with a small amount of hexane, remove the solvent on rotary evaporator, and weight out the amount of the final compound ( Figure 6).

Part 3: Synthesis of 11-hydroxyundec-2-enal
Timing: 1.5 h These steps describe the synthesis of 11-hydroxyundec-2-enal by cross-metathesis of 9-decen-1-ol and crotonaldehyde using FixCatBARF as a catalyst in a microwave reactor (Scheme 3). Reaction setup should be carried out in well ventilated fume hood.  CRITICAL: Choose a fairly large stirring element, ideally neodymium one, as mixing in a microwave reactor is difficult. Efficient mixing is necessary to form sufficiently small droplets of a suspension of the substrates and the catalyst in water.
Scheme 3. Synthesis of 11-hydroxyundec-2-enal in a microwave reactor using water as a diluent Pause point: Once the temperature is reached ($2 min), the reaction will run for 1 h. The microwave reactor then cools the reaction mixture to $20 C-25 C ($10 min).

Part 4: Purification of 11-hydroxyundec-2-enal
Timing: 3 h These steps describe the purification of the reaction mixture by removing the catalyst and distilling the crude product. All these steps should be carried out in well ventilated fume hood.
19. After completion of the reaction, transfer the mixture to a 50 mL separatory funnel and separate the aqueous phase (bottom layer) from the organic one (top layer). Troubleshooting 2 and 3. 20. Place aqueous phase in a beaker. 21. Transfer the organic phase to Erlenmeyer flask. 22. Put the aqueous phase back into the separatory funnel, add 5 mL of ethyl acetate, shake the separatory funnel vigorously and, after separating the two phases, transfer them to a suitable Erlenmeyer flasks (Figure 9).
CRITICAL: Compounds having an aldehyde group in aliphatic chain quite readily dissolve in water. That is why the extraction step should be repeated at least five times or until no product is present in the water phase. Transfer of the product to organic layer can be monitored using thin layer chromatography (silica gel, hexane:ethyl acetate, 9:1). Note: The mixture of crude product and SnatchCat can be stored for 24 h in the freezer at approximately À20 C.
26. Add drying agent portion wise (MgSO 4 or Na 2 SO 4 ), gently mix it and filter into a round bottom flask. Troubleshooting 4. 27. Remove $95% of the solvent with a rotary evaporator (35 C, 130 mbar, $10 min). 28. Prepare a silica gel pad ($10 cm length, 3 cm diameter) and place the crude product on top, washing the round bottom flask with a small amount of hexane ( Figure 10). 29. Carefully wash the product off the silica gel pad with 10%-30% solution of ethyl acetate in hexane under vacuum filtration.
CRITICAL: Greenish residue of the catalyst should remain on top of the silica gel pad after filtration. Any catalyst residues left in the crude product can cause its isomerization or decomposition.
30. Remove the solvent on a rotary evaporator (35 C, 130 mbar, $15 min). 31. Place the crude product in a small round bottom flask. 32. Purify the crude product by distillation ( Figure 11): a. set up the parameter on Kugel Rohr distillation apparatus to distill out the unreacted starting material: 200 C, 50 rpm and 5-6 mbars on vacuum pump; troubleshooting 5. b. collect the first fraction in the bulb; c. place new bulb in the apparatus, raise the temperature to 250 C-260 C, and collect the pure product; d. leave a higher boiling yellow residue in the round bottom flask.
Optional: Kugel Rohr apparatus can be replaced by standard laboratory glassware for distillation under vacuum.

EXPECTED OUTCOMES
In summary, the described protocols allow for efficient synthesis and isolation of 9-chloro-2methylnon-4-ene and 11-hydroxyundec-2-enal in high yields. In particular, harmful and toxic chlorinated or aromatic solvents can be replaced by an environmentally friendly solvent system: water with a small amount of ethyl acetate as a co-solvent. Accordingly, the proposed method does not require moisture and oxygen free conditions to perform the metathesis reaction, since water can serve as a convenient diluent of reactants. Moreover, a relatively low loading of ruthenium catalyst was necessary for full conversion of starting materials under mild conditions. In contrast to previously reported protocols, surfactant or specially designed catalysts are not necessary in these studies. Therefore, the developed method extends known protocols for aqueous metathesis methodology by utilizing microwave assistance and mechanical emulsification to increase the performance of the studied transformations in water. Consequently, we believe that the presented protocols can find broad applications in chemical synthesis, as the method is characterized by a low amount of waste produced, a low loading of the catalyst, and a high selectivity.

LIMITATIONS
The scope of substrates in this protocol is limited mainly to the lipophilic non-ionic compounds, as for the water soluble substrates metathesis reactions were not effective. Moreover, in such cases, isolation of the final product might be difficult.

TROUBLESHOOTING Problem 1
Step 2f: During the addition of the catalyst in solid form, some of the powder may settle on the sides of the vial and reactor.

Potential solution
Use the prepared ethyl acetate to wash the catalyst off the glass.

Problem 2
Steps 3 and 19: Organic and aqueous layers can be difficult to separate because of stable emulsion formation. Potential solution Add small amount of NaCl and shake mixture gently in the separatory funnel. This should help in layers separation. Brine can be also added.

Problem 3
Steps 3 and 19: During extraction, both layers have an intense, dark color and it is difficult to notice the phase division.

Potential solution
Use a flashlight to illuminate the separatory funnel on the side. Refracting rays of light will make it easier to find the interface between aqueous and organic layer.

Problem 4
Steps 9 and 26: After the extraction step, the organic layer is not clear, but cloudy and obscure.

Potential solution
The organic layer is not sufficiently dehydrated. The amount of drying agent depends on the amount of water remaining in the organic layer after the extraction step. When the drying agent stops clumping, there is enough of it.

Problem 5
Steps 15a and 32a: The vacuum achieved in the distillation system is not low enough.

Potential solution
Check that all joints are perfectly matched, and that all ground glass connections are properly lubricated with hydrocarbon based grease. If the vacuum is still not good enough despite the well-complexed distillation system, recalculate the boiling point values using the pressure-temperature nomograph.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anna Kajetanowicz (a.kajetanowicz@uw.edu.pl).

Materials availability
This study did not generate new unique reagents. All reagents used in this study were commercially available and used without further purification.

Data and code availability
All data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.